Project Summary Sperm-egg compatibility is essential to the evolutionary success of any sexually reproducing organism, yet reproductive proteins are often some of the most rapidly evolving genes in any taxa. In threespine stickleback fish (Gasterosteus aculeatus), reproductive isolation is present in many recently derived populations throughout the Northern Hemisphere, but the precise biochemical mechanisms driving this isolation are unknown. Stickleback are classic examples of molecular adaptation and speciation, but reproductive proteins are probable candidates as yet unexplored in this model evolutionary system. We hypothesize that reproductive proteins secreted by stickleback egg and sperm will display signatures of rapid Darwinian evolution, and their divergence may be involved in reproductive isolation and speciation. In Aim 1, we will identify stickleback sperm-egg recognition proteins using tandem mass spectrometry. In Aim 2, we will determine the contribution of population-level genomic differences between stickleback species pairs to reproductive isolation. The proposed research is innovative in that prior to our work, the consensus in the field has been that behavioral explanations underlie observed stickleback reproductive isolation; however, our work has found evidence for rapidly evolving stickleback egg proteins, indicating sperm competition and potentially other forms of pre-zygotic mating isolation are present in sticklebacks, challenging the current consensus in the field.